Endotoxin is a lipopolysaccharide present in a cell wall of a Gram-negative bacterium and is the most typical pyrogen. If a transfusion, a medicine for injection, blood or the like contaminated with the endotoxin enters into a human body, the endotoxin may induce severe side effects such as fever and shock. Therefore, it is required to manage the above-mentioned medicines so that they are not contaminated with endotoxin.
By the way, a hemocyte extract of limulus (hereinafter, also referred to as “limulus amoebocyte lysate (LAL)”) contains serine protease that is an enzyme activated by endotoxin. When LAL reacts with endotoxin, a coagulogen present in LAL is hydrolyzed into a coagulin by an enzyme cascade by the serine protease activated according to the amount of endotoxin, and the coagulin is associated to form an insoluble gel. With the use of this property of LAL, it is possible to detect endotoxin with a high sensitivity.
Furthermore, β-D-glucan is a polysaccharide that constitutes a cell membrane characteristic of a fungus. Measurement of β-D-glucan is effective, for example, for screening of infectious diseases due to a variety of fungi including not only fungi that are frequently observed in general clinical practices, such as Candida, Aspergillus, and Cryptococcus, but also rare fungi.
Also in the measurement of β-D-glucan, by using the property of the limulus amoebocyte lysate to coagulate (coagulate to form a gel) by β-D-glucan, the β-D-glucan can be detected with a high sensitivity.
As a method for detecting the presence of or measuring the concentration of a physiologically active substance of biological origin (hereinafter, also referred to as a predetermined physiologically active substance) such as endotoxin and β-D-glucan by a limulus hemocyte extract, there is a turbidimetric method in which a liquid mixture of a sample for detection or concentration measurement of a predetermined physiologically active substance (hereinafter, simply referred to as a “measurement of predetermined physiologically active substance”) and LAL is left standing and the turbidity of the sample due to the gel formation by a reaction between LAL and the predetermined physiologically active substance is measured over time and analyzed.
In the case of measuring the predetermined physiologically active substance with the above turbidimetric method, a liquid mixture of the measurement sample and the LAL is generated in a dry-heat sterilized measurement glass cell. Then, the gelation of the liquid mixture is optically measured from the outside. However, the turbidimetric method may take a very long time for gelation of LAL particularly in a sample with a low concentration of the predetermined physiologically active substance. Thus, an method which is capable of measuring the predetermined physiologically active substance within a short time has been desired.
In contrast, laser light scattering particle counting method has been proposed. In the laser light scattering particle counting method, a liquid mixture of a measurement sample and LAL is stirred using, for example, a magnetic stirring bar to generate fine gel particles and the presence of the predetermined physiologically active substance in the sample can be determined within a short time from the intensity of a laser light scattered by gel particles or the intensity of light passing through the liquid mixture (hereinafter, this method is also simply referred to as a light scattering method). On the other hand, a stirring turbidimetric method has been also proposed. This method is one form of the turbidimetric method, where a reaction is accelerated by stirring a measurement sample to unify the state of gelation in the liquid mixture. These methods are different in that both the turbidimetric method and the stirring turbidimetric method detect an optical transmittance, while the light scattering method detects generated particles. However, the determination in any of these methods is based on a threshold method that counts a time until the intensity of light transmitted from the liquid mixture or the number of particles calculated from the intensity or the number of peaks of scattered light exceeds a threshold.
Furthermore, there is a method in which a synthetic substrate for clotting enzyme added is previously placed in a sample and a phenomenon of coloring, fluorescence generation, or light generation of the synthetic substrate decomposed by the clotting enzyme is then determined. The method using coloring has been widely used as a colorimetric method and as one of important measurement procedures in a quantitative method of the predetermined physiologically active substance.
Among the above measurement methods, immediately after initiation of the measurement, a phenomenon in which a decrease in intensity of transmitted light is observed in the turbidimetric method and the stirring turbidimetric method, and a phenomenon in which an increase in intensity or the number of peaks of scattered light is observed in the light scattering method, without depending on the reaction of LAL (hereinafter, also referred to as a limulus reaction) with the predetermined physiologically active substance (hereinafter, this phenomenon is also referred to as progressive decrease/increase). This progressive decrease/increase affects a time until the intensity of transmitted light or the number of particles calculated from the intensity or the number of peaks of scattered light exceeds a threshold in the above measurement methods. Therefore, a decrease in measurement accuracy of the above measurement methods may occur. In the above measurement method, the lower the concentration of the predetermined physiologically active substance, the longer the measurement time until the intensity of transmitted light, or the number of particles obtained from the intensity or the number of peaks of scattered light exceeds a threshold. Thus, the lower the concentration of the predetermined physiologically active substance, the more the measurement tends to be affected by the progressive decrease/increase. In some cases, therefore, reaction-starting times, from which gelation or aggregation has started, have not been evaluated correctly.
As described above, a threshold method, a differentiation method, and the like have been used as means for determining gelation or coloring. The threshold method defines a reaction-starting time as a time point at which a physical quantity to be varied due to gelation or coloring becomes not less than a predetermined threshold set in advance or a time point at which it exceeds the threshold (hereinafter, simply referred to as a threshold-passing time point). The differentiation method is based on the degree of variation in optical transmittance or absorbance in a given period of time. It has been known that, when the threshold method is used, the relationship between the amount of a predetermined physiologically active substance in a sample and the reaction-starting time becomes a linear relationship with a negative slope in double logarithm. In addition, a time-varying curve of a physical quantity, such as optical transmittance or absorbance, to be varied due to gelation or coloring can be approximated to a logistic curve. Therefore, a very slow change is observed when it reacts with the predetermined physiologically active substance of a low concentration. In contrast, a steep change is observed when it reacts with the predetermined physiologically active substance of a high concentration. Therefore, when the same threshold is applied to both the reactions to determine a reaction-starting time, the threshold method has an inconvenience in that a measurement time for a low-concentration sample is prolonged.
On the other hand, in the differentiation method that calculates a variation in optical transmittance or absorbance, these variations and the concentration of the predetermined physiologically active substance subjected to the reaction linearly correlate with each other. However, the linear relationship is only limited to within a narrow range of concentrations. Thus, the measurement cannot be simultaneously performed at high and low concentrations.
In order to solve these problems, an area method using an area of time curve of optical transmittance or absorbance has been proposed. The area method records an area value of each time and determines a time point when the value becomes a predetermined threshold or more or exceeds the threshold as a reaction-starting time (detection time) of the predetermined physiologically active substance. As described above, however, “progressive decrease/increase”, in which optical transmittance and absorbance are actually changed at a constant rate independently of the LAL reaction, may be observed. FIG. 21 is a diagram illustrating an exemplary variation in optical transmittance over time by an endotoxin reaction. During the period of about 18 minutes from the start of the measurement, it is found that progressive-decreasing phenomenon occurs and optical transmittance linearly decreases. In such a case, the area method causes a linear increase in area value independently of the LAL reaction. Thus, in some cases, the predetermined physiologically active substance has not been correctly measured.